Therapeutic murine monoclonal antibody

ABSTRACT

The present invention relates to a monoclonal antibody which is broadly reactive with all normal human peripheral blood mononuclear cells and granulocyte, and to the hybridoma cell line which produces this monoclonal antibody. This monoclonal antibody is designated WM-65 and the hybridoma cell line is designated F56-1D5 (ECACC 89033001). This monoclonal antibody reacts with a previously unrecognized human leucocyte surface membrane antigen. The relative molecular mass of the antigen recognized by WM-65 is approximately 40-50 Kilodaltons.

FIELD OF THE INVENTION

The present invention relates to a new hybridoma cell line and to themonoclonal antibody produced by this hybridoma cell line This monoclonalantibody reacts with a novel human leucocyte surface membrane antigenwith wide distribution within the haemopoietic system.

BACKGROUND OF THE INVENTION

Since it was shown by Kohler & Milstein (Nature Vol. 256, 495-497, 1975)that it was possible to fuse mouse myeloma cells with spleen cells fromimmunized mice and thereby product a continuous cell line which producesa homogeneous (monoclonal) antibody, extensive attention has beenfocused on the production of these hybrid cell lines (hybridomas) andthe monoclonal antibodies (Mabs) produced.

The development of hybridoma technology has led to a dramaticallyimproved understanding of the antigenic molecules on the surface ofhuman leucocytes, and over the past decade, many murine monoclonalantibodies reacting with haemopoietic cell surface antigens have beendescribed. Following the three International Workshops on HumanLeucocyte Differentiation Antigen, the majority of these antibodies havebeen grouped into Clusters of Differentiation (CD), and have been shownto react with restricted differentiation-lineage or maturation stagemembrane antigens on lymphoid or bone marrow-derived cells (InterimReport of the 3rd Workshop Nomenclature Committee (1987) in A.J.McMichael, Ed. Leucocyte Typing III. White cell differentiation antigen.Oxford University Press, PP 949-950).

A few antibodies have been shown to have broader haemopoietic cellularreactivity. The best characterised are the antibodies fitting into CD 45or CD 45R, which identify a family of antigens with molecular weightsaround 200 Kilodaltons expressed on virtually all human leucocytes(Pizzolo et al., Cancer, 1980, 46, 2640-2647; Dalchau et al., 1980,European Journal of Immunology, 10, 737-744). Other unclustered Mabswith non-lineage reactivity include PHM-1, CAMPATH-1, HuLyM3, whichappear to identify separate and unique antigens (Becker et al., 1981,Pathology, 13, 669-680; Hale et al., 1983, Blood, 62, 873-882; Vaughanet al., Transplantation, 36, 446-450).

SUMMARY OF THE INVENTION

In a first aspect, the present invention consists in a mouse monoclonalantibody of class IgG₁ produced by a hybridoma formed by a fusion ofcells from a mouse myeloma line and spleen cells from a mouse previouslyimmunized with the T cell line HSB-2 and T-CLL cells, the monoclonalantibody being characterised in that it reacts with a leucocyte surfacemembrane antigen of a relative molecular weight of approximately 40-50Kilodaltons which is expressed on over 90% of normal human peripheralblood mononuclear cells and granulocytes, but not on normal platelets orerythrocytes.

It is preferred that the monoclonal antibody is further characterised inthat it:

a) reacts with approximately 90% of normal human thymocytes;

b) reacts with human B cell leukemia lines RAji, Daudi and Bristol 8;

c) reacts with human T cell leukemia lines T-All, MOLT-4, CEM, HSBO2;

d) reacts with the Myeloid cell line K562;

e) reacts with pre-B cell lines NALM-6, Reh, KM3;

f) reacts with approximately 95% of mitogen activated human lymphocytes;

g) is unreactive with the cell lines IPMI-8402, U937, Rc2a, and H160;and

h) is unreactive with platelets or erythrocytes.

In a second aspect the present invention consists in an IgG₁ monoclonalantibody-producing hybridoma cell line formed by fusion of cells from amouse myeloma line and spleen cells from a mouse previously immunizedwith the T cell line HSB-2 and T-CLL cells, the monoclonal antibodyproduced being characterised in that it reacts with a leucocyte surfacemembrane antigen of a relative molecular weight of approximately 40-50Kilodaltons which is expressed on over 90% of normal human peripheralblood mononuclear cells and granulocytes, but not on platelets orerythrocytes.

It is preferred that the monoclonal antibody produced is furthercharacterised in that it:

a) reacts with approximately 90% of normal human thymocytes;

b) reacts with human B cell leukemia lines RAji, Daudi and Bristol.8;

c) reacts with human T cell leukemia lines T-All, MOLT-4, CEM, HSB.2;

d) reacts with the Myeloid cell line K562;

e) reacts with pre-B cell lines NALM-6, Reh, KM3;

f) reacts with approximately 95% of mitogen activated human lymphocytes;

g) is unreactive with the cell lines IPMI-8402, U937, Rc2a, and H160;and

h) is unreactive with platelets or erythrocytes.

The monoclonal antibody of the first aspect of the present invention hasbeen designated WM-65, and the hybridoma cell line of the second aspectof the present invention has been designated F56-1D5. This hybridomacell line was deposited with the European Collection of Animal CellCultures (ECACC), PHLS Centre for Applied Microbiology and Research,Porton Down, Salisbury, Wilts., United Kingdom on Mar. 30, 1989 and wasaccorded accession number 89033001.

The disclosure of this deposit is herein incorporated by way ofcross-reference. The monoclonal antibody of the first aspect of thepresent invention will hereafter be referred to as WM-65 and thehybridoma cell line of the second aspect of the present invention willhereafter be referred to as F56-1D5.

DETAILED DESCRIPTION OF THE INVENTION

The hybridoma cell line F56-1D5 was produced using the techniquedescribed in detail below, and the monoclonal antibody WM-65 produced bythis hybridoma cell line was tested using the techniques describedbelow.

BRIEF DESCRIPTION OF DRAWINGS

In order that the nature of the present invention may be more clearlyunderstood preferred forms thereof will be described with reference tothe accompanying drawings in which:

FIG. 1 shows the flow cytometric evaluation of WM-65 reactivity withnormal peripheral blood mononuclear cells. Log green fluorescence isindicated on the horizontal scale and log red fluorescence on thevertical scale. In FIG. 1A, cells were labelled with negative controlMabs directly conjugated with FITC or PE. In FIGS. 1B, 1C and 1D cellswere labelled with WM-65 directly conjugated with FITC, and CD-19 Mab(B4-PE; FIG. 1B), CD-3 Mab (OKT-3-PE; FIG. 1C), or CD-11c Mab (LeuM5.PE; FIG. 1D) directly conjugated with Phycoerythrin. The resultsillustrate that virtually all CD-19⁺, CD-3⁺, and CD-11c⁺ cells arelabelled by WM-65 (indicated in quadrant II in each FIG.).

FIG. 2 shows representative immunofluorescence reactivity patterns ofWM-65 with cases of AML(FIG. 2A), and common ALL(FIG. 2B). Staining wasevaluated in a flow cytometer with log fluorescence intensity displayedon the horizontal axis and cell numbers of the vertical axis. Heavytracing indicates the fluorescence IgG₁ negative control MAB, and thelight tracing is WM-65 staining

FIG. 3 shows the autoradiagraph of immunoprecipitation and SDS-PAGEexperiment on ¹²⁵ I-labelled leucocytes. Lane A, IgG₁ negative controlMab; Lane B, WM-65 immunoprecipitation. Position of molecular weightmarkers are indicated on the left. A broad band in the range of 40-50Kilodaltons is seen in lane B.

MATERIALS AND METHODS Immunization and Hybridoma Production

An 8 week old female BALB/c mouse was injected intraperitoneally with10⁷ cells of the human T-leukemia cell line HSB-2. Five days later 5×10⁶peripheral blood leucocytes from a patient with a T cell form of chroniclymphocytic leukemia (T-CLL) were injected intraperitoneally. Twentydays later a further 8×10⁶ T-CLL lines were injected Three days laterthe animal was sacrificed and the spleen removed. Splenic lymphocyteswere fused with the mouse myeloma cell line P3-NS1-Ag3, using amodification of the method described by Kohler and Milstein (Nature,1975, 26, 495-497; and Fazekas de St. Groth et al, 1980, Journal ofImmunological Methods, 35, 1-21). Following fusion, cells were platedout in DULBECCO'S MINIMAL ESSENTIAL MEDIUM (DMEM) a tissue culturemedium containing foetal calf serum (FCS: Flow Laboratories),L-glutamine, sodium pyruvate, antibiotics andhypoxanthine-aminopterin-thymidine (HAT; Flow Labs.), into 96 wellmicrotitre plates (Linbro).

Culture supernatants from wells containing hybridomas were tested forreactivity on peripheral blood mononuclear cells. One hybridoma,F56-1D5, was selected, and cloned three times by the limiting dilutionmethod Monoclonal antibody was produced by injecting 10⁷ hybridoma cellsintraperitoneally into pristane-primed mice, and collecting asciticfluid.

Preparation of Cells for Antibody Characterization

Mononuclear cells were isolated from heparinized peripheral bloodobtained from healthy volunteers by centrifugation on FICOLL-HYPAQUE(Pharmacia), a centrifugation medium, while granulocytes were preparedfrom the same source using MONO-POLY (Pharmacia), a centrifugationmedium. Tonsil and thymic lymphocytes were obtained from fresh surgicalsamples. The tissue was cut and teased in medium to produce a singlecell suspension. The cells were washed and tested immediately, orcryopreserved until required.

Leukaemic cells were obtained from heparinized bone marrow or peripheralblood diagnostic samples, and separated on FICOLL-HYPAQUE. Cells wereeither used fresh or cryopreserved until required. Leukaemic cell lineswere grown in RPMI 1640 (Flow Labs.) containing FCS, L-glutamine andantibiotics.

Immunofluorescence Staining and Flow Cytometry

Peripheral blood leucocytes were prepared for immunofluorescent stainingas described previously (Bradstock et al, 1985, Pathology, 17, 392-399).Briefly, 1-2×10⁶ cells were reacted at 20° C. with a saturatingconcentration of Mab for 10 minutes, then washed in phosphate bufferedsaline containing 0.1% sodium azide (PBSA), followed by incubation withsheep anti-mouse antiserum conjugated to fluorescein isothiocyanate(SAM-FITC; Silenus) for a further 10 minutes at room temperature. Forcharacterization of subsets of normal peripheral blood mononuclearcells, two colour direct immunofluorescence was used. Leucocytes werestained with WM-65 directly conjugated to FITC, and with Mabs to CD-3(OKT-3 PE), CD-11c (Leu M5-PE), or CD-19 (B4 PE) all directly conjugatedwith Phycoerythrin. After completion of immunofluorescence staining,cells were then washed again, and reactivity determined using a FACS 440cytometer (Becton Dickinson, Calif.). An isotype-matched Mab unreactivewith human cells was used as a negative control.

Complement Mediated Cytoxicity

The lytic ability of the Mab was tested in a complement-mediatedcytoxicity assay as previously described. Briefly, 10⁶ peripheral bloodmononuclear cells were incubated with a saturating concentration of Mabfor 15 minutes at room temperature. This was followed by the addition ofan equal volume of rabbit serum (Pelfreez), and further incubation at37° C. for 45 minutes. A Mab (W6-32; Sera Labs.) reactive with humanClass 1 MHC antigens was used as a positive control Cell viability wasdetermined by Trypan blue exclusion using an inverted microscope.

Reactivity with Bone Marrow Progenitor Cells

Normal bone marrow was obtained with the informed consent of normalvolunteers undergoing harvest for allogeneic bone marrowtransplantation. Mononuclear cells were separated on FICOLL-HYPAQUE,washed, then reacted with a saturating solution of Mab or with theappropriate negative control for 10 minutes at 20° C. After washing withPBS the cells were incubated with SAM-FITC, for a further 10 minutes at20° C., washed again, and then sorted into fluorescence positive andnegative populations in a FACS440 cell sorter. Leucocytes from eachpopulation were plated at 1×10⁵ viable cells in quadruplicate in 0.3%agar containing 25% FCS in 35 mm Petri dishes, with a 0.5% agarunderlayer containing 1×10⁶ irradiated normal peripheral bloodmononuclear cells as a feeder layer. Plates were incubated in 5% CO₂ inair at 37° C., and examined at day 12 using an inverted microscope.Colonies were scored as groups of more than 40 cells.

Immunoprecipitation and Electrophoresis

Antigenic molecular weight determination was performed as previouslydescribed (Bradstock et al, 1985, Pathology, 17, 392-399). Peripheralblood mononuclear cells were surface-labelled with ¹²⁵ I using thelactoperoxidase method Cells were disrupted using TRITON-X 100, adetergents and centrifuged to remove cytoskeletal material Theradiolabelled lysate was precleared overnight using PANSORBIN(Pharmacia), an absorption medium. The cell lysate was reacted with Mabfor 2 hours at 4° C. followed by a further 30 minutes incubation withgoat antimouse IgG (Cappell). The complex was absorbed onto PABNSORBINand resuspended in non-reducing electrophoresis buffer. Samples werereduced by the addition of 15 ul mercaptoethanol Samples, together withmolecular weight markers (Biorad), were run on a 5-15% gradientpolyacrylamide gel overnight. The gels were stained with CoomassieBrilliant Blue, dried and autoradiographs performed usingHYPERFILM(Amersham), an X-ray film at -70° C.

Tissue Section Staining

Fresh tissue biopsies were snap frozen in isopentane and stored inliquid nitrogen Cryostat sections 6 to 8 microns thick were air driedonto poly-L-Lysine (Sigma) treated slides, fixed in acetone at -10° C.for 5 minutes, and washed with 3% hydrogen peroxide in 0.05 MTris-HCl/phosphate buffered saline, pH 7.6, for 5 minutes to blockendogenous peroxidase. Tissue sections were then washed in Tris-bufferedsaline for 15 minutes. Prior to immunohistochemical staining, sectionswere incubated with diluted normal horse serum (Vectastain ABC kit, No.PK4002) for 5 minutes to reduce non-specific binding of the secondaryantibody. Mab (or the isotype-matched negative control) in the form of1:10 dilution of culture supernatant was then added to the sections, andincubated for one hour in a humidified chamber on a platform rocker. Theslides were washed in Tris-PBS and incubated with an appropriatedilution of biotinylated horse anti-mouse antibody (Vectastain) for 20minutes. After further washing in Tris-PBS, sections were incubated for30 minutes with avidin-horseradish peroxidase complex (Vectastain),washed again, then developed for 4 minutes in DAB solution (0.03% 3,3'diamino benzidine tetra-hydrochloride, Fluka, Switzerland) and 0.2 mMimidazole (Sigma) in PBSA. After further washing in water for 5 minutes,sections were counterstained with Mayer's haematoxylin and blueingsolution, before dehydration in absolute alcohol, clearing in xylol andmounting.

RESULTS Cellular Reactivity of WM-65

The hybridoma selected, F56-1D5, secreted a murine IgG₁ Mab which wasdesignated WM-65. Its reactivity with both normal and leukaemichaemopoietic cells is detailed in Tables 1 & 2. WM-65 reacted with over90% of peripheral blood mononuclear cells (FIG. 1) and granulocytes, butnot with normal platelets or erythrocytes (Table 1). It reacted withvirtually all thymocytes and tonsil lymphocytes, as well as with themajority of normal bone marrow mononuclear cells. However, in myeloidprogenitor assays, WM-65 reacted with only a minority (mean value 13.6%,range 2-34%, 5 experiments) of normal CFU_(GM) (Table 3). The reactivityof WM-65 with PHA-stimulated T lymphoblasts was equivalent to that seenon resting normal T lymphocytes. Based on dye exclusion studies, WM-65was incapable of lysing mononuclear cells in the presence of rabbitserum.

The reactivity of WM-65 with cell lines and leukaemic cells is alsoshown in Table 2. WM-65 reacted with all pre-B and B cell lines testedas well as 4/5 T cell lines, but with only 1/4 myeloid cell lines. WM-65showed extensive reactivity with leukaemic cells, including 8/8 cases ofcommon acute lymphoblastic leukaemia (c-ALL), 4/4 B-chronic lymphaticleukaemia (B-CLL) and 2/2 hairy cell leukaemia (HCL), together with13/13 acute myeloid

                  TABLE 1                                                         ______________________________________                                        REACTIVITY OF WM-65 WITH                                                      NORMAL HAEMOPOIETIC CELLS                                                     CELL TYPE         PERCENTAGE POSITIVE.sup.a                                   ______________________________________                                        Peripheral blood mononuclear                                                  cells                                                                         Total             96.6 +/- 2.8                                                                              (n = 7)                                         T lymphocytes (CD-3.sup.+)                                                                      96.6 +/- 4.0                                                                              (n = 4)                                         B lymphocytes (CD-20.sup.+)                                                                     97.5 +/- 0.9                                                                              (n = 4)                                         monocytes (CD-11c.sup.+)                                                                        97.5 +/- 0.5                                                                              (n = 4)                                         Granulocytes      96.2 +/- 1.5                                                                              (n = 5)                                         Platelets          1          (n = 5)                                         Erythrocytes       1          (n = 5)                                         Thymocytes        91.5 +/- 0.5                                                                              (n = 2)                                         Tonsil lymphocytes                                                                              90.5 +/- 5.5                                                                              (n = 2)                                         Bone marrow mononuclear cells                                                                   63.2 +/- 10.6                                                                             (n = 5)                                         ______________________________________                                         Footnotes                                                                     .sup.a Percentage of cells +/- 1 standard deviation positive above            negative control by immunofluorescence.                                  

                  TABLE 2                                                         ______________________________________                                        REACTIVITY OF WM-65 WITH                                                      LEUKAEMIC CELLS AND CELL LINES                                                                      PERCENTAGE                                              CELL TYPE             POSITIVE                                                ______________________________________                                        (A)  Leukaemias.sup.a                                                              C-ALL                .sup. 8/8.sup.b                                          T-ALL                2/5                                                      AML                  13/13                                                    CML                  5/6                                                      B-CLL                4/4                                                      T-CLL                1/1                                                      Hairy cell leukaemia 2/2                                                      PLL                  1/1                                                 (B)  Leukaemic cell lines                                                          B cell                                                                        Daudi                .sup. 98.sup.c                                           Raji                 98                                                       Bristol 8            98                                                       T cell                                                                        T-ALL-1              90                                                       MOLT-4               98                                                       RPMI-8402             1                                                       CEM                  98                                                       HSB-2                98                                                       Pre-B                                                                         NALM-6               98                                                       Reh                  72                                                       KM-3                 55                                                       Myeloid                                                                       K-562                98                                                       HL-60                 5                                                       RC-2A                 5                                                       U937                  5                                                  (C)  Non-haemopoietic tumor cell lines                                             Melanoma             0/1                                                      Squamous cell carcinoma                                                                            0/2                                                      Fallopian carcinoma  0/1                                                      Ovarian carcinoma    0/4                                                      Cervical carcinoma   0/2                                                      Neuroblastoma        0/1                                                      Breast carcinoma     0/1                                                 ______________________________________                                         Footnotes                                                                     .sup.a Abbreviations used: CALL, common form of acute lymphoblastic           leukaemia; TALL, T cell form of ALL; AML, acute myeloid leukaemia; CML,       chronic myeloid leukaemia; BCLL, B cell form of chronic lymphatic             leukaemia; TCLL, T cell form of CLL; PLL, prolymphocytic leukaemia.           .sup.b Number of cases considered to be positive with WM65, with 20% of       cells fluorescent above negative control, over total number tested.           .sup.c Percentage of cells fluorescent above negative control for each        cell line.                                                               

                  TABLE 3                                                         ______________________________________                                        REACTIVITY OF WM-65 WITH NORMAL BONE                                          MARROW MYELOID PROGENITOR CELLS.sup.a                                                    NUMBER OF CFU.sub.GM                                               FRACTION     EXPERIMENT 1 EXPERIMENT 2                                        ______________________________________                                        UNSEPARATED  125 (100)    266 (100)                                           WM-65.sup.+  9 (5)        174 (34)                                            WM-65.sup.-  164 (95)     332 (66)                                            ______________________________________                                         Footnote                                                                      .sup.a Bone marrow cells incubated with WM65 and SAMFITC, and passed          through a FACS 440 cell sorter, either unsorted, or sorted into               fluorescencepositive and negative populations. Two representative             experiments are shown CFU.sub.GM values were adjusted to 10.sup.5 cells       per plate, with assays being performed in quadruplicate, and mean values      tabulated above. Values in parentheses indicate the proportion of             CFU.sub.GM in each population. Proportion of bone marrow cells positive       for WM65 was 47% in Experiment 1, and 60% in Experiment 2.               

leukaemia (AML) and 5/6 chronic myeloid leukaemia (CML) (FIG. 2). Thepercentage of cells positively stained by WM-65 in each case of acuteleukaemia was generally high, with a mean of 71.7% of AML blasts (range35.3-96.5) labelled, and mean 75.6% (range 21.8-90.7) ALL cells positivefor WM-65. All non-haemopoietic cell lines tested were negative (Table2).

Immunochemical Characterization of the Antigen Recognised by WM-65

SDS-polyacrylamide gel electrophoresis of ¹²⁵ I-surface-labelledperipheral blood mononuclear cells revealed that WM-65immunoprecipitated a broad band with an apparent molecular weightbetween 40-50KD under both reduced and non-reduced conditions (FIG. 3).

Non-Haemopoietic Tissue Reactivity of WM-65

Frozen sections of normal tissues were evaluated for WM-65 reactivity byimmunoperoxidase using an avidin-biotin-peroxidase complex. WM-65 wasreactive with virtually all lymphoid cells and macrophages in both lymphnode and tonsil, but showed no staining of salivary gland, lung, kidney,skeletal muscle and heart, apart from occasional lymphoid cells andmacrophages.

WM-65 reacts with a novel human leucocyte differentiation antigen withwide distribution within the haemopoietic system. On immunoprecipitationand polyacrylamide gel electrophoresis, the antigen can be identified asa single broad band of molecular weight in the range of 40-50Kilodaltons, indicating that it is likely to be a single polypeptidewith heavy but variable glycosylation. This antigen is expressed onvirtually all nucleated bone marrow-derived cells and lymphocytes, butwas not detectable on non-haemopoietic cells.

It is interesting to note that although a high proportion of bone marrowmononuclear cells were reactive with WM-65, these did not include themajority of myeloid progenitors as judged by CFU_(GM) assays, indicatingthat the antigen detected by WM-65 may be maturation linked, andexpressed only as lymphocytes mature from earlier marrow stem cells.However, WM-65 did react with immature malignant haemopoietic cells,including leukaemic cell lines as well as cases of both ALL and AML.

Overall the data indicate that WM-65 reacts with an antigen notpreviously described. A number of leucocyte antigens with broadnon-lineage specific distribution have been described previously(Pizzolo et al., 1980, Cancer, 46 2640-2647; Dalchau et al., 1980,European Journal of Immunology, 10, 737-744; Becker et al., 1981,Pathology, 13, 669-680; Hale et al., 1983, Blood, 62, 873-882; Vaughanet al., Transplantation, 36, 446-450). Differences in both molecularweight and cellular distribution distinguish these previously describedantigens from the antigen recognised by WM-65 (Table 4). Class I MHCantigens have a molecular weight of 47KD, and are associated with thecell membrane of B₂ -microglobulin (12KD) (Strominger, 1980, In Progressin Immunology Vol. 4, London Academic Press, p 539). Although the HLAheavy chain is closely similar to that of the WM-65 antigen, nomolecular weight band comparable to the B-2M was seen onimmunoprecipitation with WM-65. In addition, class I antigens arepresent on platelets but not on the B cell line Daudi, a pattern ofexpression opposite to that seen with WM-65 (Barnstable et al., 1978,Cell, 14, 9-20; Brown et al. 1979, European Journal of Immunology, 9,272-275). The T200 or leucocyte common antigens (CD-45) have a molecularweight in the range of 180-220KD (Cobbold et al., 1987, In LeucocyteTyping III. White Cell Differentiation Antigens Oxford University Press,P788-803). Although the cellular reactivity patterns of CD-45 Mabs andWM-65 are very similar (Table 3), the molecular weights of the antigensrecognised are

                                      TABLE 4                                     __________________________________________________________________________    COMPARISON OF WM-65 WITH OTHER NON-LINEAGE                                    SPECIFIC ANTIBODIES.sup.a                                                     CELL LINE                                                                            WM-65                                                                             CD45 CLASS II                                                                            PHM-1                                                                             HuLyM3                                                                             CAMPATH                                        __________________________________________________________________________    PBL    .sup. +.sup.b                                                                     +    +     +   +    +                                              Granulocytes                                                                         +   +    +     +   +    +/-                                            Patelets                                                                             -   -    +     -   -    -                                              HSB-2  +   +    +     +   +    +/-                                            U937   -   +    +     +   +    -                                              KM3    +   +    +     +   +    +                                              K562   +   +    +     +   -    +                                              Reh    +   +    +     +   +    +                                              MW (K.D).sup.c                                                                       40-50                                                                             220-180                                                                            47,12 180,62                                                                            47   23-30                                          __________________________________________________________________________     Footnotes                                                                     .sup.a All cellular reactivities determined in the inventors' laboratory.     .sup.b +: reactivity 20%, +/-: reactivity 15-20%,                             -: reactivity 15% by indirect immunofluorescence and flow cytometry.           .sup.c Antigen molecular weights quoted from original references.       

clearly different.

Three other Mabs with similar reactivity have been described. PHM₁reacts with lymphocytes, granulocytes and several cell lines, andimmunoprecipitates an antigen of molecular weight 180/62KD (Becker etal., 1981, Pathology, 13, 669-680). Aside from this report of molecularweight, the major property distinguishing PHM-1 from WM.65 is that theformer reacts with the U937 cell line, which is unreactive with WM-65.The pan leucocyte Mab HuLyM3 immunoprecipitates an antigen of 47KDmolecular weight, with a cellular distribution closely similar to thatof WM-65 (Vaughan et al., Transplantation, 36, 446-450). However, HuLyM3does not react with the myeloid cell line K562, which is moderatelystrongly labelled by WM.65, indicating that the two Mabs identifydifferent antigens. Finally, CAMPATH-1 can be distinguished from all theabove in that it does not react with granulocytes to any significantdegree, and precipitates a broadly glycoslated band of 23-30 KD (Hale etal., 1983, Blood, 62, 873-882; Cobbald et al., 1987, In Leucocyte TypingIII. White Cell Differentiation Antigens. Oxford University Press,P788-803).

The above data indicates that the 40-50 Kilodalton molecule identifiedby WM-65 Mab is a newly recognised leucocyte surface membrane antigen.Although its restricted distribution to the haemopoietic family of cellssuggests some functional significance, no physiological role for themolecule has yet been investigated. The major practical feature ofinterest with this Mab is its extensive reactivity with acute andchronic leukaemias of both myeloid and lymphoid types. This is anunusual property for a monoclonal antibody, and raises the interestingpossibility that the antibody may be useful in the treatment ofhaematological malignancies. This is heightened by the fact that WM-65has comparatively little reactivity with bone marrow progenitor cells,and might therefore allow therapy to be selectively targeted tomalignant haemopoietic cells. Although the antibody is notcomplement-fixing, it may have clinical application if it was conjugatedto a toxin, drug or radioisotope. Preliminary studies to link thebiological toxin ricin to WM-65 are underway to explore thispossibility.

We claim:
 1. A mouse monoclonal antibody produced by the hydridoma cellline designated F56-1D5 (ECACC 89033001).
 2. A hybridoma cell line inwhich the cell line is designated F56-1D5 (ECACC 89033001).